Detection of patterns of interactions among proteins can prove of great conceptual and diagnostic value in malignancy and other diseases, but efficient methods have been lacking to characterize interactions both in research and diagnostics. We will take advantage of a unique set of tools developed in our lab to now establish methods to: 1) Characterize interactions among large sets of proteins directly in patient samples using the proximity ligation technique and a recently established paired-tag array read-out, for interaction biomarker discovery. 2) Develop and validate smaller-scale diagnostic assays of interacting protein molecules using a combination of array read-out and a novel in situ interaction analysis that we have also recently established. The procedure we will use in the characterization phase involves immobilization of in situ crosslinked interacting proteins to a solid support. A series of antibodies specific for proteins of interest, conjugated with oligonucleotides including antigen-specific tag sequences, are allowed to bind to proteins in the biological samples in so-called proximity ligation reactions. The presence of interacting proteins results in co-localization of specific pairs of antibodies, which in turn brings their attached oligonucleotides in close proximity, allowing these to be joined by enzymatic ligation. The resulting DNA molecules are combinations of tags that reflect the identity of the detected pairs of interacting proteins. The ligated molecules are amplified, restriction digested, and sorted on an array containing all possible pairs of tag sequences for the proteins under investigation. Paired-tag sequences properly hybridized to the arrayed molecules can be circularized, and the oligonucleotides immobilized on the array next serve as primers for rolling circle reaction where only correctly ligated, circular molecules are amplified. The resulting rolling circle products contain tandem repeats of generic detection sequence, permitting hybridization of a fluorescent probe for array detection. The method will be established and applied to study interactions among members of the Smad protein family, intracellular effectors of the transforming growth factor (TGF)-[unreadable] signaling pathway that play well-documented roles in both tumor suppression and tumor progression. In the second phase, which will temporally overlap with the first, assays adapted for diagnostic use will be developed, and interacting pairs of proteins will be validated as markers using this assay, and also by studies in tissue sections from patient samples, with the help of so-called proximity ligation in situ assays. In these assays, proximal binding of pairs of antibodies with attached oligonucleotides result in the formation of circularized DNA strands for localized rolling-circle replication reactions that permit even single interacting pairs of proteins to give rise to clearly detectable signals. The proposed procedures allow endogenous protein complexes to be observed without the requirement for cloning or transfecting exogenous components, permitting dynamic interaction networks to be investigated in normal, precancerous, and cancerous cells. Furthermore, any pair-wise interactions among a given set of proteins can be monitored, and the paired-tag system is scalable and transferable to study different pathways. The procedures and results of this project will establish protein interaction events as markers for tumor diagnostics, and for evaluation of drug treatment.